In all cells, proteins are synthesized by ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) substrates to translate messenger RNA (mRNA). In recent years, tremendous progress has been made in elucidating the structure of the ribosome in the absence and presence of substrates and various translation factors. Despite this wealth of structural information, several important questions about translation elongation and the role of rRNA in this process remain open. Aim 1 of this proposal is to determine the role of 16S rRNA in decoding. A number of mutations in 16S rRNA that increase misreading in vivo will be characterized in vitro. The data obtained may reveal how signaling between the 30S A site and elongation factor EF-Tu is mediated. Aim 2 of this proposal is to determine the molecular basis of ribosomal unlocking, which limits the rate of translocation. Nine inter-subunit bridge mutations and four 30S neck mutations will be thoroughly characterized with respect to factor-independent and factor-dependent translocation in vitro. The data obtained will identify those ribosome- ribosome contacts contributing to the energy barrier for translocation and may help uncover the molecular basis of unlocking. Ribosomes are a main target of antibiotics, and defects in translation are associated with a growing number of inherited human diseases and cancers. Insight gained by this project may lead to the development of novel antimicrobial drugs and/or treatments for one or more hereditary diseases.